The predominant CD 1d-restricted cell population in the mouse system is the Va14 NKT cell, a cell-type expressing a semi-invariant Va14-Ja281/VB8 TCR and a panoply of receptors of the NK lineage. Va14 NKT cells can be specifically stained by tetramers of CD 1d loaded with their glycolipid antigen a-GalCer and represent 3 to 30 percent of the T lymphocytes in various tissues. They have functions that partially overlap with those of NK cells, mediated by the secretion of Th1 cytokine and by their cytolytic properties, or are unique, mediated by their rapid release of IL4. Thus, NKT cells and NK cells exemplify both common and unique principles governing the development and function of cellular elements of the Innate Immune system. Our long term objective is to understand the rules governing the development and selection of the CD 1d-restricted aB T cell population, especially with regards to the expression of NK lineage receptors. The specific aims of this continuation application are: 1- To identify the developmental stages of cells expressing the canonical Va14 TCR in normal and Va14 Tg mice. CD 1d/aGalCer tetramers will be used to follow their thymic and post-thymic development, especially with regards to the acquisition of the memory and NK phenotype, cytokine secretion profile and migratory pattern. 2- To identify the defective stages of Va14 T cell development in NKT deficient mice, including natural (SJL, NOD, 129) or mutant (IL-15, fyn) NKT deficient strains. 3- To determine whether the unique pattern of CD1d expression is required for thymic selection and differentiation of Va14 NKT cells. By redirecting the expression of CD1d into a classical MHC pattern with an Ea-CD 1d transgene, we will test the impact of its distribution on the selection and differentiation of cells expressing Va14 and other CD1d-restricted TCRS. 4- To determine the function of the CD1d endosomal targeting motif in the development and selection of Va14 NKT cells. The deletion of this motif markedly decreases CD1d autorecognition by mature Va 14 NKT cells in vitro. The deletion will therefore be introduced into the germline through homologous recombination to assess its impact on the various stages of NKT development in vivo.